Process for making quinolone compounds

ABSTRACT

The present invention relates to the field of synthesizing anti-infective compounds. More particularly, the invention relates to synthesizing a family of quinolone compounds useful as anti-infective agents. The invention includes a process for preparing a quinolone compound wherein less than about 0.40% of dimeric impurity of the quinolone is produced.

This application is a U.S. National Phase Application under 35 U.S.C.§371 of International Patent Application No. PCT/US2009/005276 filedSep. 23, 2009, which claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/194,083 filed on Sep. 24, 2008, each ofwhich is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of synthesizinganti-infective compounds. More particularly, the invention relates tosynthesizing a family of quinolone compounds useful as anti-infectiveagents. The invention includes a process for preparing a quinolonecompound wherein less than about 0.40% of dimeric impurity of thequinolone is produced.

BACKGROUND

Since the discovery of penicillin in the 1920s and streptomycin in the1940s, many new compounds have been discovered or specifically designedfor use as antibiotic agents. It was once believed that infectiousdiseases could be completely controlled or eradicated with the use ofsuch therapeutic agents. Resistant strains of Gram-positive bacteriasuch as methicillin-resistant staphylocci, penicillin-resistantstreptococci, and vancomycin-resistant enterococci have developed, whichcan cause serious and even fatal results for patients infected with suchresistant bacteria. Bacteria that are resistant to macrolideantibiotics, i.e., antibiotics based on a 14- to 16-membered lactonering, have developed. Also, resistant strains of Gram-negative bacteriasuch as H. influenzae and M. catarrhalis have been identified. See,e.g., F. D. Lowry, “Antimicrobial Resistance: The Example ofStaphylococcus aureus,” J. Clin. Invest., 2003, 111(9), 1265-1273; andGold, H. S, and Moellering, R. C., Jr., “Antimicrobial-Drug Resistance,”N. Engl. J. Med., 1996, 335, 1445-53.

Despite the problem of increasing antibiotic resistance, no new majorclasses of antibiotics have been developed for clinical use since theapproval in the United States in 2000 of the oxazolidinonering-containing antibiotic,N-[[(5S)-3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methylacetamide, which is known as linezolid and is sold under the tradenameZyvox® (see compound A). See, R. C. Moellering, Jr., “Linezolid: TheFirst Oxazolidinone Antimicrobial,” Annals of Internal Medicine, 2003,138(2), 135-142.

Linezolid was approved for use as an anti-bacterial agent active againstGram-positive organisms. Unfortunately, linezolid-resistant strains oforganisms are already being reported. See, Tsiodras et al., Lancet,2001, 358, 207; Gonzales et al., Lancet, 2001, 357, 1179; Zurenko etal., Proceedings Of The 39^(th) Annual Interscience Conference OnAntibacterial Agents And Chemotherapy (ICAAC); San Francisco, Calif.,USA, (Sep. 26-29, 1999).

Notwithstanding the foregoing, there is an ongoing need for newanti-infective agents and for methods of making them.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a prediction profiler for the amount of dimeric impurity 4when ethyl acetate (EtOAc) is the solvent. This was based on the initialdesign of experiments. The center line of each plot shows the predictedvalues and the two lines flanking the center line represent theapproximately ±95 percent confidence levels. The horizontal dotted linesignifies a dimer 4 level of 0.235094 percent. The vertical dotted linessignify the variables for 1.05 equivalents of N-chlorosuccinimide (NCS),3.5 mole percent sulfuric acid, 17° C., 0.05 percent water content inthe solvent and an NCS addition rate of 0.1 volume per minute. The 95percent confidence limit is ±0.040991 for the values indicated in thepreceding sentence.

FIG. 2 shows the impact of H₂SO₄ and time on the levels of dimericimpurity 4.

FIG. 3 shows the worst case scenario in prediction profiler for theamount of dimeric impurity 4 for robustness of DoE, i.e. for the seconddesign of experiments. The center line of each plot shows the predictedvalues and the two lines flanking the center line represent theapproximately ±95 percent confidence levels. The horizontal dotted linesignfies a dimer 4 level of 0.1045 percent. The vertical dotted linessignify the variables for 1.04 equivalents of N-chlorosuccinicmide(NCS), 21° C., an NCS addition rate of 30 minutes, and 0.8 mole percentsulfuric acid. The 95 percent confidence limit is +0.009339 for thevalues indicated in the preceding sentence.

FIG. 4 shows an initial design of experiments experimental table.

FIG. 5 a shows an actual by prediction plot for the initial design ofexperiments of FIG. 4.

FIG. 5 b shows a summary of fit for the initial design of experiments ofFIG. 4.

FIG. 5 c shows an analysis of variance for the initial design ofexperiments of FIG. 4.

FIG. 5 d shows parameter estimates for the initial design of experimentsof FIG. 4.

FIG. 5 e show a residual by predicted plot for the initial design ofexperiments of FIG. 4.

FIG. 5 f shows sorted parameter estimates for the initial design ofexperiments of FIG. 4.

FIG. 6 a shows a prediction profiler for the initial design ofexperiments of FIG. 4.

FIG. 6 b show interaction profiles for the initial design of experimentsof FIG. 4.

FIG. 7 shows a robustness design of experiments experimental table for asecond design of experiments.

FIG. 8 a shows an actual by predicted plot for the second design ofexperiments of FIG. 7.

FIG. 8 b shows a summary of fit for the second design of experiments ofFIG. 7.

FIG. 8 c shows an analysis of variance for the second design ofexperiments of FIG. 7.

FIG. 8 d shows a lack of fit for the second design of experiments ofFIG. 7.

FIG. 8 e shows parameter estimates for the second design of experimentsof FIG. 7.

FIG. 8 f shows a residual by predicted plot for the second design ofexperiments of FIG. 7.

FIG. 8 g shows a prediction profiler for the second design ofexperiments of FIG. 7.

SUMMARY OF THE INVENTION

The present invention relates to the field of synthesizinganti-infective compounds. More particularly, the invention relates tosynthesizing a family of quinolone compounds useful as anti-infectiveagents.

The present invention relates to a process for preparing a quinolonecompound comprising the step of reacting a des-chloro quinolone compoundor a pharmaceutically acceptable salt or ester thereof with achlorinating agent and an acid, wherein less than about 0.40% on an areapercent basis as quantified by analytical HPLC of dimeric impurity ofthe quinolone is produced.

In other embodiments the present invention relates to a process wherethe des-chloro quinolone compound is1-(6-amino-3,5-difluoropyridin-2-yl)-6-fluoro-7-(3-hydroxy-azetidin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid or a pharmaceutically acceptable salt or ester thereof, thequinolone compound is1-(6-amino-3,5-difluoropyridin-2-yl)-8-chloro-6-fluoro-7-(3-hydroxy-azetidin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid or a pharmaceutically acceptable salt or ester thereof.

In other embodiments the present invention relates to a process wherethe dimeric impurity is compound1-amino-3-(azetidin-3-yloxy)-propan-2-ol-bis(N,N′-quinolone carboxylicacid), or a pharmaceutically acceptable salt or ester thereof. In otherembodiments the present invention relates to a process where the dimericimpurity is a mono-ester. In other embodiments the present inventionrelates to a process where the dimeric impurity is a di-ester.

In other embodiments the present invention relates to a process wherethe chlorinating agent is N-chlorosuccinimide.

In other embodiments the present invention relates to a process wherethe acid is selected from the group consisting of sulfuric acid,hydrochloric acid, hydrobromic acid, phosphoric acid, trifluoroaceticacid, triflic acid, methanesulfonic acid, p-toluenesulfonic acid orperchloric acid, and mixtures thereof.

In other embodiments the present invention relates to a process wherethe acid is sulfuric acid.

In other embodiments the present invention relates to a process wherethe reaction is run at a temperature from about 0° C. to about 30° C.

In other embodiments the present invention relates to a process wherethe reaction is run at a temperature from about 15° C. to about 25° C.

In other embodiments the present invention relates to a process wherethe reaction is run at a temperature from about 13° C. to about 21° C.

In other embodiments the present invention relates to a process wherethe molar ratio of N-chlorosuccinimide to des-chloro quinolone isgreater than about 1.

In other embodiments the present invention relates to a process wherethe molar ratio of N-chlorosuccinimide to des-chloro quinolone is fromabout 1.05 to 1.2.

In other embodiments the present invention relates to a process wherethe molar ratio of N-chlorosuccinimide to des-chloro quinolone is fromabout 1.04 about 1.07.

In other embodiments the present invention relates to a process wherethe molar ratio of sulfuric acid to des-chloro quinolone is from about0.005 to about 0.05.

In other embodiments the present invention relates to a process wherethe molar ratio of sulfuric acid to des-chloro quinolone is from about0.007 to about 0.02.

In other embodiments the present invention relates to a process wherethe molar ratio of sulfuric acid to des-chloro quinolone is from about0.008 to about 0.012.

In other embodiments the present invention relates to a process where anacetate ester as a solvent.

In other embodiments the present invention relates to a process wherethe acetate ester is selected from the group consisting of methylacetate, ethyl acetate, and mixtures thereof.

In other embodiments the present invention relates to a process wheresaid acetate ester is methyl acetate.

In other embodiments the present invention relates to a processcomprising the further step of reacting the quinolone compound with abase.

In other embodiments the present invention relates to a process wherethe base is a hydroxide base.

In other embodiments the present invention relates to a process wherethe hydroxide base is selected from the group consisting of sodiumhydroxide, potassium hydroxide, lithium hydroxide, barium hydroxide, andmixtures thereof.

In other embodiments the present invention relates to a process wherethe hydroxide base is potassium hydroxide.

In other embodiments the present invention relates to a process using amixture of C1-C6 alcohol and water as a solvent.

In other embodiments the present invention relates to a process wherethe C1-C6 alcohol is isopropanol.

In other embodiments the present invention relates to a process wherethe process is a commercial scale process.

In other embodiments the present invention relates to a compositioncomprising a quinolone compound or salt or ester thereof having lessthan about 0.40% of dimeric impurity of the quinolone compound.

In other embodiments the present invention relates to a compositionwherein the quinolone compound is1-(6-amino-3,5-difluoropyridin-2-yl)-8-chloro-6-fluoro-7-(3-hydroxy-azetidin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid or a pharmaceutically acceptable salt or ester thereof.

In other embodiments the present invention relates to a compositionwhere the dimeric impurity is1-amino-3-(azetidin-3-yloxy)-propan-2-ol-bis(N,N ′-quinolone carboxylicacid), or a pharmaceutically acceptable salt or ester thereof.

In other embodiments the present invention relates to a compositionwhere the composition is a commercial scale composition.

In other embodiments the present invention relates to a process or acomposition where the dimeric impurity is less than about 0.35%.

In other embodiments the present invention relates to a process or acomposition where the dimeric impurity is less than about 0.30%.

In other embodiments the present invention relates to a process or acomposition where the dimeric impurity is less than about 0.25%.

In other embodiments the present invention relates to a process or acomposition where the dimeric impurity is less than about 0.20%.

In other embodiments the present invention relates to a process or acomposition where the dimeric impurity is less than about 0.15%.

In other embodiments the present invention relates to a process or acomposition where the dimeric impurity is less than about 0.10%.

In other embodiments the present invention relates to a process or acomposition where the dimeric impurity is less than about 0.05%.

In other embodiments the present invention relates to a process orcomposition where the dimeric impurity is less than about 0.04%.

In other embodiments the present invention relates to a process orcomposition where the dimeric impurity is less than about 0.03%.

In other embodiments the present invention relates to a process orcomposition where the dimeric impurity is less than about 0.02%.

In other embodiments the present invention relates to a process orcomposition where said dimeric impurity is less than about 0.01%.

DETAILED DESCRIPTION OF THE INVENTION

Quinolones

The processes and compositions of the present invention comprise aquinolone compound.

Quinolone compounds, such as pyridonecarboxylic acid derivatives, usefulherein are described, including their synthesis, formulation, and use,in U.S. Pat. No. 6,156,903, to Yazaki et al., issued Dec. 5, 2000 andits certificates of correction of Nov. 13, 2001 and Dec. 11, 2001; U.S.Pat. No. 6,133,284, to Yazaki et al., issued Oct. 17, 2000; U.S. Pat.No. 5,998,436, to Yazaki et al., issued Dec. 7, 1999 and itscertificates of correction of Jan. 23, 2001, Oct. 30, 2001, and Dec. 17,2002; PCT Application No. WO 2006/110815, to Abbott Laboratories,published Oct. 19, 2006; PCT Application No. WO 2006/042034, to AbbottLaboratories, published Apr. 20, 2006, PCT Application No. WO2006/015194, to Abbott Laboratories, published Feb. 9, 2006; PCTApplication No. WO 01/34595, to Wakunaga Pharmaceutical Co., Ltd.,published May 17, 2001; and PCT Application No. WO 97/11068, to WakunagaPharmaceutical Co., Ltd., published Mar. 27, 1997.

Pyridonecarboxylic acid derivatives of the present invention includecompounds corresponding to the following structure (PyridonecarboxylicAcid Derivative 1)

wherein R¹ represents a hydrogen atom or a carboxyl protective group; R²represents a hydroxyl group, a lower alkoxy group, or a substituted orunsubstituted amino group; R³ represents a hydrogen atom or a halogenatom; R⁴ represents a hydrogen atom or a halogen atom; R⁵ represents ahalogen atom or an optionally substituted saturated cyclic amino group;R⁶ represents a hydrogen atom, a halogen atom, a nitro group, or anoptionally protected amino group; X, Y and Z may be the same ordifferent and respectively represent a nitrogen atom, CH or CR⁷ (whereinR⁷ represents a lower alkyl group, a halogen atom, or a cyano group),with the proviso that at least one of X, Y and Z represent a nitrogenatom, and W represents a nitrogen atom or CR⁸ (wherein R⁸ represents ahydrogen atom, a halogen atom, or a lower alkyl group), and with theproviso that when R¹ represents a hydrogen atom, R² represents an aminogroup, R³ and R⁴ represent a fluorine atom, R⁶ represents a hydrogenatom, X represents a nitrogen atom, Y represents CR⁷ (wherein R⁷represents a fluorine atom), Z represents CH, and W is CR⁸ (wherein R⁸represents a chlorine atom), then R⁵ is not a 3-hydroxyazetidine-1-ylgroup; or a pharmaceutically acceptable salt, ester, or prodrug thereof.

As described in the foregoing paragraph, when R¹ is a carboxylprotective group, it may be any carboxylate ester residue which cleavesrelatively easily to generate the corresponding free carboxyl group.Exemplary carboxyl protective groups include those which may beeliminated by hydrolysis, catalytic reduction, and other treatmentsunder mild conditions such as lower alkyl groups such as methyl group,ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butylgroup, t-butyl group, pentyl group, hexyl group, and heptyl group; loweralkenyl groups such as vinyl group, allyl group, 1-propenyl group,butenyl group, pentenyl group, hexenyl group, and heptenyl group;aralkyl groups such as benzyl group; and aryl groups such as phenylgroup and naphthyl group; and those which may be readily eliminated inthe body such as lower alkanoyloxy lower alkyl groups such asacetoxymethyl group and pivaloyloxymethyl group; lower alkoxycarbonyloxylower alkyl group such as methoxycarbonyloxymethyl group and1-ethoxycarbonyloxyethyl group; lower alkoxymethyl group such asmethoxymethyl group; lactonyl group such as phthalidyl; di-loweralkylamino lower alkyl group such as 1-dimethylaminoethyl group; and(5-methyl-2-oxo-1,3-dioxole-4-yl)methyl group.

It is noted that the substituents R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, A,J¹, J², J³, W, X, Y, Z, e, f, and g are defined herein for conveniencewith respect to the chemical structure for the pyridonecarboxylic acidderivatives.

In other embodiments, the present invention relates to a process forpreparing a pyridonecarboxylic acid derivative of structurePyridonecarboxylic Acid Derivative 1, wherein W is CR⁸, wherein R⁸represents a hydrogen atom, a halogen atom, or a lower alkyl group.

In other embodiments, the present invention relates to a process forpreparing a pyridonecarboxylic acid derivative of structurePyridonecarboxylic Acid Derivative 1, wherein R⁵ is a group representedby the following formula (a) or (b):

wherein A represents an oxygen atom, sulfur atom or NR⁹ (wherein R⁹represents hydrogen atom or a lower alkyl group), e represents a numberfrom 3 to 5, f represents a number from 1 to 3, g represents a numberfrom 0 to 2, J¹ , J² and J³, which may be the same or different from oneanother, represent a hydrogen atom, hydroxyl group, lower alkyl group,amino lower alkyl group, amino group, lower alkylamino group, loweralkoxy group, or a halogen atom.

In other embodiments, the present invention relates to a process forpreparing a pyridonecarboxylic acid derivative of structurePyridonecarboxylic Acid Derivative 1, wherein R⁵ is a group representedby formula (a).

In other embodiments, the present invention relates to a process forpreparing a pyridonecarboxylic acid derivative of structurePyridonecarboxylic Acid Derivative 1, wherein e in the formula (a) is 3or 4.

In other embodiments, the present invention relates to a process forpreparing a pyridonecarboxylic acid derivative of structurePyridonecarboxylic Acid Derivative 1, wherein R¹ is a hydrogen atom; R²is an amino group, lower alkylamino group, or a di-lower alkylaminogroup; R³ is a halogen atom; R⁴ is a halogen atom; R⁶ is hydrogen atom;X is a nitrogen atom; Y and Z are CH or CR⁷ (wherein R⁷ is a lower alkylgroup or a halogen atom); and W is CR⁸ (wherein R⁸ is a halogen atom ora lower alkyl group).

In other embodiments, the present invention relates to a process forpreparing a pyridonecarboxylic acid derivative of structurePyridonecarboxylic Acid Derivative 1, wherein R² is amino group; R³ isfluorine atom; R⁴ is a fluorine atom; Y is CF; Z is CH; W is CR⁸(wherein R⁸ is a chlorine atom, bromine atom or a methyl group), and ein formula (a) is 3.

In other embodiments, the present invention relates to a process forpreparing a pyridonecarboxylic acid, wherein said pyridonecarboxylicacid corresponds to the following structure:

or a pharmaceutically acceptable salt, ester, or prodrug thereof. Thisforegoing pyridonecarboxylic acid is also known by the publiclydisclosed code names Abbott Laboratories ABT-492, WakunagaPharmaceutical Co., Ltd. WQ 3034, Rib-X Pharmaceuticals, Inc., RX-3341,the USAN delafloxacin, and also by the chemical names1-(6-amino-3,5-difluoro-2-pyridinyl)-8-chloro-6-fluoro-1,4-dihydro-7-(3-hydroxy-1-azetidinyl)-4-oxo-3-quinolinecarboxylicacid,1-(6-amino-3,5-difluoro-2-pyridinyl)-8-chloro-6-fluoro-1,4-dihydro-7-(3-hydroxyazetidin-1-yl)-4-oxo-3-quinolinecarboxylicacid, 3-quinolinecarboxylic acid,1-(6-amino-3,5-difluoro-2-pyridinyl)-8-chloro-6-fluoro-1,4-dihydro-7-(3-hydroxy-1-azetidinyl)-4-oxo,and1-(6-amino-3,5-difluoropyridin-2-yl)-8-chloro-6-fluoro-7-(3-hydroxyazetidin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid. This carboxylic acid form of the compound corresponds to the CASregistry number 189279-58-1. Furthermore, WO 2006/042034, cited abovediscloses the D-glucitol salt of this compound [D-glucitol1-(6-amino-3,5-difluoro-2-pyridinyl)-8-chloro-6-fluoro-1,4-dihydro-7-(3-hydroxy-1-azetidinyl)-4-oxo-3-quinolinecarboxylate(salt)] and the trihydrate of the D-glucitol salt of this compound[D-glucitol1-(6-amino-3,5-difluoro-2-pyridinyl)-8-chloro-6-fluoro-1,4-dihydro-7-(3-hydroxy-1-azetidinyl)-4-oxo-3-quinolinecarboxylatetrihydrate (salt)]. The D-glucitol salt and the D-glucitol salttrihydrate correspond to the CAS registry numbers 352458-37-8 and883105-02-0, respectively. D-glucitol corresponds to the CAS registrynumber 6284-40-8. WO 2006/042034 also discloses a crystalline form ofthe D-glucitol salt characterized when measured at about 25° C. withCu-Ka radiation, by the powder diffraction pattern shown in FIG. 1 (seeWO 2006/042034) and a crystalline form of the D-glucitol salt trihydratewhen measured at about 25° C. with Cu-Ka radiation, by the powderdiffraction pattern shown in FIG. 2 (see WO 2006/042034). TheseD-glucitol salts are useful in the present invention. Also, see A. R.Haight et al., “Synthesis of the Quinolone ABT-492: Crystallizations forOptimal Processing”, Organic Process Research & Development (2006),10(4), 751-756.

The terms “commercial scale process” and “commercial scale composition”refer to a process and composition, respectively, which is run orproduced as a single batch of at least about 100 grams.

Identification and Suppression of a Dimer Impurity in the Development ofDelafloxacin

See, Hanselmann, R., et al., “Identification and Suppression of a DimerImpurity in the Development of Delafloxacin”, Organic Process Research &Development, vol. 13, pages 54-59 (2009).

Delafloxacin is a 6-fluoroquinolone antibiotic which is underdevelopment at Rib-X Pharmaceuticals, Inc. During initial scale up runsto prepare delafloxacin, up to 0.43% of a new impurity arose in thepenultimate chlorination step. This was identified as a dimeric adductof delafloxacin. Subsequent application of design of experiments (DoE's)led to the identification of the factors responsible for this impurity.Implementation of the knowledge gained from the DoE's reproduciblyenabled the suppression of this impurity to acceptable levels.

Antimicrobial resistance in the community and hospital settings has beena growing public health concern due to the continuing emergence ofmultidrug resistant bacterial strains. See (a) Cosgrove, S. E.; Carmeli,Y. Clin. Infect. Dis. 2003, 36, 1433. (b) Seybold, U.; Kourbatova, E.V.; Johnson, J. G.; Halvosa, S. J.; Wang, Y. F.; King, M. D.; Ray, S.M.; Blumberg, H. M. Clin. Infect. Dis. 2006, 42, 647. and (c) Tenover,F. C.; McDougal, L. K.; Goering, R. V.; Kiligore, G.; Projan, S. J.;Patel, J. B.; Dunman, P. M. J. Clin. Microbiol. 2006, 44, 108.

Methicillin-resistant Staphylococcus aureus (MRSA) ranks as the mostfrequently isolated pathogen in hospital intensive care units in theUnited States and the incidence of MRSA occurrence increased from 35.9%in 1992 to 64.4% in 2003. See Klevens, R. M.; Edwards, J. R.; Tenover,F. C.; McDonald, L. C.; Horan, T.; Gaynes, R. Clin. Infect. Dis. 2006,42, 389.

Since the introduction of nalidixic acid nearly 40 years ago, quinoloneantibiotics have occupied a prominent place in the array of antibiotics.6-Fluoroquinolones such as ciprofloxacin have especially gained anexpanding role in the treatment of infections, due to their broadspectrum of application. See (a) Bush, K. Clin. Microbiol. Infect. 2004,10 (Suppl. 4), 10. and (b) Emmerson, A. M.; Jones, A. M. J. Antimicrob.Chemother. 2003, 51 (Suppl. S1), 13.

Delafloxacin is a 6-fluoroquinolone antibiotic with excellentantibacterial activity against gram-positive organisms, including bothmethicillin-susceptible S aureus and MRSA. It is currently undergoingphase II clinical trials. Delafloxacin was initially developed byWakunaga Pharmaceuticals and Abbott Laboratories and was subsequentlylicensed by Rib-X Pharmaceuticals, Inc.

The synthesis of delafloxacin initially underwent development by AbbottLaboratories (Scheme 1) and a key step in this is a selectivechlorination on the 8-position of the functionalized quinolone 1 (thedes-chloro quinolone). See, (a) Haight, A. R.; Ariman, S. Z.; Barnes, D.M.; Benz, N. J.; Gueffier, F. X.; Henry, R. F.; Hsu, M. C.; Lee, E. C.;Morin, L.; Pearl, K. B.; Peterson, M. J.; Plata, D. J.; Willcox, D. R.Org. Process Res. Dev. 2006, 4, 751. and (b) Barnes, D. M.; Christesen,A. C.; Engstrom, K. M.; Haight, A. R.; Hsu, M. C.; Lee, E. C.; Peterson,M. J.; Plata, D. J.; Raje, P. S.; Stoner, E. J.; Tedrow, J. S.; Wagaw,S. Org. Process Res. Dev. 2006, 4, 803.

In this process a solution of 1 in a mixture of methyl acetate (MeOAc)and ethyl acetate is chlorinated using NCS in the presence of 3.5 mole %of H2SO4, affording 2. This is followed by solvent exchange andsaponification with KOH to yield 3. Delafloxacin is obtained after saltformation with N-methyl-D-glucamine.

Despite initial success in implementing this process, we encountereddifficulties upon scale up of this step in that up to 0.43 area % of anew impurity was detected by HPLC at RRT 1.60 after isolation of 3.Additionally, this new impurity turned out to be difficult to purgeduring the final salt formation. Thus, we decided to initiate a study toidentify this impurity, understand how it was being formed and suppressits generation.Results and Discussion

Despite numerous attempts to isolate this new impurity by preparativeHPLC, we were unable to do so and only the molecular weight wasestablished by HPLC-MS as 880 Da. The measured molecular weight of thisimpurity is exactly double of the acid 3, which suggested a dimericderivative of this compound. Close examination of the purity profile ofthe substrate of the chlorination reaction did not lead to the detectionof any impurity that could similarly be assigned a dimeric structure andits formation was therefore attributed to the chlorination—hydrolysissequence. A number of potential dimeric adducts that could arise in thisstep were considered, including 4, which would result from cleavage ofthe azetidine moiety in one unit of 3 and reacting with the hydroxylgroup of a second. In order to investigate this possibility further, weembarked on the synthesis of 4.

Retrosynthetically (Scheme 2), molecule 4 is readily disconnected into asuitably protected amino alcohol 5 and quinolone 6; the latter is aknown compound. Fragment 5 can be prepared from commercially availableazetidin-3-ol hydrochloride 7. See, (a) Yazaki, A.; Niino, Y.; Ohshita,Y.; Hirao, Y.; Amano, H.; Hayashi, N.; Kuramoto, Y. PCT Int. Appl. WO9711068, 1997. CAN: 126, 305587. and (b) Yazaki, A.; Aoki, S. PCT Int.Appl. WO 2001034595, 2001. CAN: 134, 366811.

The synthesis thus commenced from 7, in which the nitrogen was protectedas a benzyl carbamate to yield 8 in quantitative yield. This adduct wasalkylated with racemic epichlorohydrin to give 9 in 84% yield. Epoxideopening of 9 with ammonia gave 10, which was condensed withoutpurification with 6 to yield 11 in 83% overall yield. Deprotection ofthe Cbz group under hydrogenation conditions gave 12 in 93% yield. Asecond condensation with 6 resulted in formation of the dimeric compound13 in 71% yield. After saponification the presumed impurity 4 wasobtained in 98% yield.

With synthetic 4 at hand, the unknown impurity in a contaminated batchof delafloxacin was compared with synthesized 4 via spiking experimentsand comparison by HPLC-MS and HPLC-UV. To our delight, synthetic 4matched unambiguously with the unknown impurity seen in previouslymanufactured batches of delafloxacin.

In order to understand the dynamics of the formation of impurity 4, wedecided to investigate the chlorination reaction further in a design ofexperiments (DoE) study. The following factors were chosen to beinvestigated in a DoE study of resolution IV, over ranges as specified:temperature (15-25° C.), amount of NCS (1.05-1.2 eq.), amount of H2SO4(2-5 mol %), water content in solvent (0-0.5%), solvent volume (2-3vol.), solvent (methyl acetate/ethyl acetate) and NCS addition rate(0.05-0.3 vol/min.). See, FIG. 4, FIGS. 5 a, 5 b, 5 c, 5 d, 5 e, and 5f, FIGS. 6 a and 6 b, FIG. 7, and FIGS. 8 a, 8 b, 8 c, 8 d, 8 e, 8 f,and 8 g. A total of 19 chlorination reactions were performed in aMultiMax™ reactor, available from Mettler-Toledo, Inc., 1900 PolarisParkway, Columbus, Ohio, 43240.

In each case samples from the reactions were quenched after 5 h,saponified with KOH and the crude reaction mixtures were analyzed byHPLC. In order to determine the amount of 4, the chlorinated samples 2were saponified to 3. The area % value for impurity 4 that resulted ineach case was processed and analyzed using the DoE software. Theexperimental design and analysis were conducted using JMP, Design ofExperiments, Version 7, SAS Institute Inc., Cary, N.C., 1989-2007, usinga stepwise fit followed by a standard least squares method.

An excellent correlation of R2 of 0.997 was obtained followingprocessing of the data. Of the main effects, higher amounts of NCS,lowering the temperature and faster addition of the NCS solution as wellas use of dry solvents had the most beneficial impact on suppressing theamount of impurity 4 (FIG. 1). Methyl acetate containing less than 500ppm water was used, prior to adjustment as required by the appropriateexperiment in the DoE's. Additionally, a strong interaction was observedbetween the amount of NCS and solvent, in that methyl acetate ispreferred when only a slight excess of NCS is used. In order to suppressany overchlorination of 2, 1.05 equivalents of NCS is preferred andhence methyl acetate was chosen as the solvent of choice for this step.A more detailed analysis can be found in FIG. 4, FIGS. 5 a, 5 b, 5 c, 5d, 5 e, and 5 f, FIGS. 6 a and 6 b, FIG. 7, and FIGS. 8 a, 8 b, 8 c, 8d, 8 e, 8 f, and 8 g.

From a mechanistic point of view, we postulate that impurity 4 couldarise from an initial acid catalyzed activation of the azetidine ringwhich triggers an isobutyric ester/chloride induced ring openingsequence to 16. During the subsequent saponification 16 reacts with thehydrolysis intermediate 17 or 3 to 4 (Scheme 4). Saponification andsubsequent epoxide formation of 16 prior to condensation with 3 or 17cannot be ruled out. The validity of this sequence was furtherstrengthened by a subsequent HPLC-MS analysis of a crude chlorinationreaction before saponification. In this, an impurity with a molecularweight of 574 Da, which matches 16, was detected in approximate equalamounts compared to 4 after saponification.

Based on this hypothetical mechanism, a time dependency for theformation of 4 during the chlorination process cannot be excluded, andsince the reaction time was kept constant in the DoE study, it wasdecided to evaluate this parameter independently. A chlorinationreaction was performed using 3.5% of H₂SO₄ and methyl acetate as solventat 15° C. and a sample was quenched after the reaction was deemed to becomplete. Additional samples were quenched after 2 h and 6 h, saponifiedand analyzed by HPLC. Not surprisingly, a steady increase of theimpurity 4 over time was seen. This result has an impact on controllingthe chlorination process, in that an adequate turn around time for theHPLC monitoring of this reaction would be necessary in order to minimizethe formation of 4. However, subsequent experiments showed thatdecreasing the amount of H₂SO₄ to 1% diminished the amount of impurity 4produced over time without having a significant impact on thechlorination reaction time or the quality of 3 (FIG. 2). Thus, anacceptable turn around time for the in-process control can be achievedwhen a level of 1% of H₂SO₄ is employed as catalyst.

After having established a good understanding of the critical parameterswith respect to formation of this impurity, a second DoE study wasinitiated to test the robustness of the reaction in the anticipatedprocess operating range. In this, a DoE study of resolution IV wasdesigned with the following factors undergoing variation: temperature(13-21° C.), amount of NCS (1.04-1.07 eq.), NCS addition rate (30-75min.) and H₂SO₄ (0.8-1.2 mol %). A total of 10 chlorination reactionswere performed in a MultiMax™ reactor. In each case samples werequenched and saponified after passing the in-process control. Theresulting area % of 4 was processed and analyzed using the DoE software.As anticipated, temperature, amount of NCS and H₂SO₄ had a statisticallysignificant effect on the amount of impurity 4 in the studied parameterrange. However, assuming the worst case scenario in the predictionprofiler, impurity 4 has a value of 0.11 area %±0.01%, which is wellwithin the acceptable limit that has been established from atoxicological batch of delafloxacin (FIG. 3).

Two subsequent kilo lab runs of this reaction confirmed theeffectiveness of the parameter changes and material of high quality withimpurity 4 levels of 0.07% were obtained after saponification.

In conclusion, we have successfully identified a dimer impurity whichwas detected during scale up of delafloxacin. Subsequent DoE experimentsenabled us to identify means to control this impurity to acceptablelevels in small scale as well as in kilo lab runs.

EXAMPLES Example 11-(6-Amino-3,5-difluoro-pyridin-2-yl)-8-chloro-6-fluoro-7-(3-hydroxy-azetidin-1-yl)-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid, 3, Improved procedure

To a suspension of 1 (3.1 kg, 6.15 mol) in methyl acetate (8.6 kg) wasadded a solution of H₂SO₄ (5.9 g, 62 mmol) and NCS (0.88 kg, 6.46 mol)in methyl acetate (14.4 kg) at 10-17° C. within 45 min. The solution wasstirred at 13-19° C. for 2 h, quenched with 1.6% aqueous NaHCO₃ (12.6kg) and the organic layer was washed with 11% aqueous Na₂SO₃ (7 kg). Themethyl acetate solution was solvent exchanged to 2-propanol at 50°C./vacuum, then a solution of KOH (1.1 kg, 19.7 mol) in water (24.8 kg)was added and the mixture was stirred at 55° C. for 3 h. 13% Aqueousacetic acid (2.6 kg) was added at 40° C. and the solution was seededwith 3 (27 g, 61 mmol). The suspension was stirred for 1 h at 40° C. andthen 13% aqueous acetic acid (11.7 kg) was slowly added. After stirringan additional hour at 40° C. the suspension was cooled to roomtemperature, filtered, washed with water (41 kg) and dried at 60°C./vacuum to yield 3 as yellow crystals (2.5 kg, 91%). Isolated 3 hadthe same spectroscopic properties as reported.

Example 2 1-Amino-3-(azetidin-3-yloxy)-propan-2-ol-bis(N,N′-quinolonecarboxylic acid), 4 3-Hydroxy-azetidine-1-carboxylic acid benzyl ester,8

To a solution of azetidin-3-ol hydrochloride 7 (25 g, 0.23 mol) in water(150 mL) and THF (300 mL) was added K₂CO₃ (63.1 g, 0.46 mol). Themixture was stirred for 30 min. at 20-25° C. Then benzyl chloroformate(40.9 g, 0.24 mol) was added within 30 min. at 0-5° C. followed bystirring the mixture overnight at 20-25° C. THF was removed on a rotavapat 30° C./vacuum and the mixture was extracted with ethyl acetate (2×150mL). The combined organic layer was washed with water (1×50 mL), driedover Na₂SO₄ and concentrated. The residue was purified by flash columnchromatography on silica gel, eluting with ethyl acetate-heptanes 1:1and 4:1 to yield 8 as a clear oil (47.3 g, 100%). ¹H NMR (300 MHz,CDCl₃): δ 3.72 (1H, d, J=6.2 Hz), 3.85 (2H, dd, J=9.5, 4.4 Hz), 4.17(2H, dd, J=9.5, 6.7 Hz), 4.49-4.57 (1H, m), 5.06 (2H, s), 7.31-7.38 (5H,m); 13C NMR (75 MHz, CDCl₃): δ 59.2, 61.6, 66.9, 127.9, 128.1, 128.5,136.5, 156.6; IR: (film) 3406, 1686, 1438 cm⁻¹; ES-HRMS m/z: (M++1H)Calcd. for C₁₁H₁₄NO₃ 208.0968. Found 208.0967.

3-Oxiranylmethoxy-azetidine-1-carboxylic acid benzyl ester, 9

To a solution of 8 (30 g, 0.15 mol) in DMSO (250 mL) was slowly added asolution of NaOH (9.9 g, 0.25 mol) in water (195 mL) at 15-25 oC.Epichlorohydrin (93.8 g, 1.01 mol) was added and the mixture was stirredat 20-25° C. for 24 h. The mixture was diluted with water (300 mL) andextracted with ethyl acetate (2×150 mL). The combined organic layer waswashed with water (2×50 mL), dried over Na₂SO₄ and concentrated. Theresidue was purified by flash column chromatography on silica gel,eluting with ethyl acetate-heptanes 3:2, yielding 9 as a clear oil (32.1g, 84%). 1H NMR (300 MHz, CDCl₃): δ 2.60 (1H, dd, J=4.8, 2.6 Hz), 2.81(1H, dd, J=4.9, 4.2 Hz), 3.09-3.16 (1H, m), 3.25 (1H, dd, J=11.4, 6.2Hz), 3.68 (1H, dd, J=11.5, 2.5 Hz), 3.89-3.97 (2H, m), 4.15-4.24 (2H,m), 4.29-4.37 (1H, m), 5.09 (2H, s), 7.28-7.36 (5H, m); 13C NMR (75 MHz,CDCl₃): δ 44.2, 50.4, 56.7, 56.9, 66.7, 68.6, 70.0, 128.0, 128.1, 128.5,136.6, 156.5; IR: (film) 2951, 1709, 1420 cm-1; ES-HRMS m/z: (M++1H)Calcd. for C₁₄H₁₈NO₄ 264.1230. Found 264.1230.

1-(6-Amino-3,5-difluoro-pyridin-2-yl)-7-[3-(1-benzyloxycarbonyl-azetidin-3-yloxy)-2-hydroxy-propylamino-]-8-chloro-6-fluoro-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid ethyl ester, 11

A mixture of 9 (19 g, 72.2 mmol) in conc. NH₄OH (380 mL) and 7M NH₃ inMeOH (86 mL) was stirred for 5 h at room temperature. The clear solutionwas concentrated and azeotropically dried with toluene. The residualclear oil and 6 (20 g, 48.1 mmol) were dissolved in NMP (150 mL).N,N-diisopropylethylamine (12.4 g, 96.2 mmol) was added and the solutionwas stirred at 70° C. for 3 h. The solution was poured into 1N citricacid/ice (300 mL) and extracted with ethyl acetate (2×150 mL). Thecombined organic layer was washed with water (2×100 mL), dried overNa₂SO₄ and concentrated. The residue was purified by flash columnchromatography on silica gel, eluting with ethyl acetate-heptanes 1:1followed by ethyl acetate-MeOH 95:5, yielding 11 as a yellow foam (27.1g, 83%). ¹H NMR (300 MHz, CDCl₃): δ 1.35 (3H, t, J=7.1 Hz), 3.35-3.52(4H, m), 3.62-3.77 (1H, m), 3.84-3.91 (2H, m), 3.95-4.08 (1H, m), 4.15(2H, dd, J=9.3, 6.5 Hz), 4.23-4.30 (1H, m), 4.35 (2H, q, J=7.1 Hz),4.85-5.13 (3H, br. s), 5.08 (2H, s), 7.18-7.25 (1H, m), 7.31-7.35 (5H,m), 7.99 (1H, dd, J=13.7, 3.1 Hz), 8.31 (1H, s); 13C NMR (75 MHz,CDCl₃): δ 14.4, 48.5 (d, JF=10 Hz), 56.6, 61.1, 66.9, 68.6, 69.3, 70.8,107.2, 111.5, 112.6 (d, JF=24 Hz), 113.2 (m), 120.6, 128.0, 128.1,128.5, 134.1 (d, JF=5 Hz), 134.7 (m), 136.5, 139.2 (d, JF=13 Hz), 144.9(d, JF=253 Hz), 144.4 (d, JF=13 Hz), 145.6 (dd, JF=262, 4 Hz), 149.9 (d,JF=246 Hz), 150.0, 156.5, 164.7, 172.9; IR: (KBr) 2949, 1700, 1615 cm-1;ES-HRMS m/z: (M++1H) Calcd. for C₃₁H₃₀ClF₃N₅O₇ 676.1780. Found 676.1762.

1-(6-Amino-3,5-difluoro-pyridin-2-yl)-7-[3-(azetidin-3-yloxy)-2-hydroxy-propylamino]-8-chloro-6-fluoro-4-oxo-1,4-dihydro-quinoline-3-carboxylicacid ethyl ester, 12

To a slurry of 10% Pd on carbon (2.1 g) in MeOH (20 mL) was added asolution of 11 (13.7 g, 20.3 mmol) in MeOH (230 mL). The mixture washydrogenated at 1 atm. for 1 h, filtered over Hyflo and evaporatedyielding 12 as beige crystals (10.3 g, 93%). Mp. 148-152° C.; 1H NMR(300 MHz, DMSO-d₆): δ 1.27 (3H, t, J=7.1 Hz), 3.27 (1H, d, J=5.0 Hz),3.28-3.80 (10H, m), 4.19 (1H, br. s), 4.21 (2H, q, J=7.1 Hz), 5.86 (1H,s), 6.74 (2H, s), 7.84 (1H, d, J=13.8 Hz), 7.94 (1H, dd, J=9.7, 9.0 Hz),8.43 (1H, s); 13C NMR (75 MHz, CDCl₃): δ 14.1, 48.4 (d, JF=10 Hz), 53.6,60.2, 68.4, 70.4 (d, JF=4 Hz), 72.1, 106.4 (d, JF=6 Hz), 111.0, 111.3(d, JF=23 Hz), 113.6 (dd, JF=23, 21 Hz), 118.9 (d, JF=6 Hz), 133.8 (d,JF=13 Hz), 134.2, 139.5 (d, JF=12 Hz), 143.3 (dd, JF=248, 4 Hz), 145.0(dd, JF=259, 5 Hz), 145.6 (d, JF=14 Hz), 149.3 (d, JF=245 Hz), 149.5,163.5, 171.0; IR: (KBr) 1697, 1614, 1496, 1457 cm-1; ES-HRMS m/z:(M++1H) Calcd. for C₂₃H₂₄ClF₃N₅O₅ 542.1413. Found 542.1391.

1-Amino-3-(azetidin-3-yloxy)-propan-2-ol-bis(N,N′-quinolone diester), 13

A solution of 12 (9.6 g, 17.7 mmol), 6 (7.8 g, 18.6 mmol) andN,N-diisopropylethylamine (4.6 g, 35.4 mmol) in NMP (150 mL) was stirredat 55° C. for 3 h. The solution was poured into 1N citric acid/ice (300mL) and extracted with ethyl acetate (3×100 mL). The combined organiclayer was washed with water (2×100 mL), dried over Na₂SO₄ andconcentrated. The residue was purified by flash column chromatography onsilica gel, eluting with ethyl acetate-MeOH 95:5. The obtained yellowfoam was crystallized with CH₂Cl₂-MeOH 9:1 (160 mL), yielding 13 asbeige crystals (11.8 g, 71%). Mp. 184-187° C.;

1H NMR (300 MHz, DMSO-d₆): δ 1.26 (6H, t, J=7.1 Hz), 3.29-3.48 (3H, m),3.49-3.62 (1H, m), 3.73-3.82 (1H, m), 4.12-4.30 (3H, m), 4.21 (4H, q,J=7.1 Hz), 4.52-4.65 (2H, m), 5.13-5.22 (1H, m), 5.83-5.92 (1H, m), 6.72(4H, s), 7.73 (1H, d, J=13.9 Hz), 7.82 (1H, d, J=13.9 Hz), 7.92 (1H, t,J=9.6 Hz), 7.93 (1H, t, J=8.7 Hz), 8.41 (2H, s); 13C NMR (75 MHz,CDCl₃): δ 12.3 (2×), 46.4 (d, JF=11 Hz), 58.4 (2×), 61.9 (2×), 66.7,67.3 (d, JF=4 Hz), 69.1, 103.4 (d, JF=6 Hz), 104.6 (d, JF=6 Hz), 108.7(d, JF=23 Hz), 109.2, 109.4 (d, JF=23 Hz), 109.5, 111.7 (dd, JF=25, 24Hz), 111.8 (dd, JF=25, 24 Hz), 117.1 (d, JF=7 Hz), 117.8 (d, JF=6 Hz),132.1 (dd, JF=17, 4 Hz), 132.2, 132.5, 133.5, 137.7 (d, JF=12 Hz), 139.4(d, JF=12 Hz), 141.0 (dd, JF=247, 5 Hz), 141.5 (dd, JF=248, 5 Hz), 143.0(dd, JF=259, 5 Hz), 143.3 (dd, JF=259, 5 Hz), 143.8 (2×, d, JF=15 Hz),147.5 (d, JF=245 Hz), 147.7, 147.8, 148.1 (d, JF=247 Hz), 161.7 (2×),169.1, 169.2; IR: (KBr) 1728, 1615, 1491, 1448 cm-1; ES-HRMS m/z:(M++1H) calcd. for C₄₀H₃₃Cl₂F₆N₈O₈ 937.1697. Found 937.1696.

1-Amino-3-(azetidin-3-yloxy)-propan-2-ol-bis(N,N′-quinolone carboxylicacid), 4

To a suspension of 13 (17.0 g, 18.1 mmol) in 2-propanol (75 mL) wasadded a 1N KOH solution (127 mL, 126.7 mmol). After stirring the mixtureat 55° C. for 3.5 h, the solution was cooled to 30° C. and a solution ofAcOH (12.4 g, 206.5 mmol) dissolved in water (94 mL) was added within 1h. The suspension was stirred at room temperature for 2 h, filtered,washed with water (3×40 mL) and dried at 50° C./vacuum, yielding 4 asyellow crystals (15.7 g, 98%). Mp. 198-205 oC (decomp.); ¹H NMR (300MHz, DMSO-d6): δ 3.28-3.45 (2H, m), 3.45-3.78 (2H, m), 3.79-3.88 (1H,m), 4.16-4.33 (3H, m), 4.61-4.75 (2H, m), 5.25 (1H, br. s), 6.23-6.35(1H, m), 6.76 (4H, s), 7.79 (1H, d, J=13.7 Hz), 7.90 (1H, d, J=13.8 Hz),7.93 (2H, dd, J=9.7, 2.4 Hz), 8.70 (1H, s), 8.71 (1H, s), 14.59 (2H, br.s); 13C NMR (75 MHz, CDCl₃): □ 48.1 (d, JF=11 Hz), 63.8, 68.4, 69.0 (d,JF=5 Hz), 70.6 (d, JF=6 Hz), 104.5 (d, JF=6 Hz), 105.9 (d, JF=7 Hz),107.8, 108.2, 109.8 (d, JF=23 Hz), 110.8 (d, JF=23 Hz), 113.4 (d, JF=23Hz), 113.7 (d, JF=23 Hz), 115.8 (d, JF=8 Hz), 116.6 (d, JF=8 Hz), 133.3(dd, JF=14, 3 Hz), 133.5 (dd, JF=14, 4 Hz), 134.8, 135.9, 141.0 (d,JF=12 Hz), 142.1 (d, JF=12 Hz), 142.8 (dd, JF=249, 5 Hz), 143.3 (dd,JF=249, 5 Hz), 145.1 (dd, JF=259, 5 Hz), 145.4 (dd, JF=260, 5 Hz), 145.6(2×, d, JF=15 Hz), 149.5 (d, JF=248 Hz), 150.1 (2×), 150.2 (d, JF=249Hz), 164.7, 164.8, 175.8 (d, JF=3 Hz), 175.9 (d, JF=3 Hz); IR: (KBr)1727, 1622, 1489, 1439 cm-1; ES-HRMS m/z: (M++1H) Calcd. forC₃₆H₂₅C₁₂F₆N₈O₈ 881.1071, found 881.1090.

Additional Experimental Materials

Experimental tables and analyses of the DoE studies are further providedin FIG. 4, FIGS. 5 a, 5 b, 5 c, 5 d, 5 e, and 5 f, FIGS. 6 a and 6 b,FIG. 7, and FIGS. 8 a, 8 b, 8 c, 8 d, 8 e, 8 f, and 8 g.

Formulation and Administration

The compounds of the present invention can be practiced by deliveringthe compounds of the present invention using any suitable carrier. Thedose of active compound, mode of administration and use of suitablecarrier will depend upon the intended patient or subject and thetargeted microorganism, e.g., the target bacterial organism. Theformulations, both for human medical use and veterinary use, ofcompounds according to the present invention typically include suchcompounds in association with a pharmaceutically acceptable carrier.

The carrier should be “acceptable” in the sense of being compatible withcompounds of the present invention and not deleterious to the recipient.Pharmaceutically acceptable carriers, in this regard, are intended toinclude any and all solvents, dispersion media, coatings, absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds (identified or designed according to the invention and/orknown in the art) also can be incorporated into the compositions. Theformulations can conveniently be presented in dosage unit form and canbe prepared by any of the methods well known in the art ofpharmacy/microbiology. In general, some formulations are prepared bybringing the compound into association with a liquid carrier or a finelydivided solid carrier or both, and then, if necessary, shaping theproduct into the desired formulation.

A pharmaceutical composition of the invention should be formulated to becompatible with its intended route of administration. Solutions orsuspensions can include the following components: a sterile diluent suchas water, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

A wide variety of formulations and administration methods, including,e.g., intravenous formulations and administration methods can be foundin S. K. Niazi, ed., Handbook of Pharmaceutical Formulations, Vols. 1-6[Vol. 1 Compressed Solid Products, Vol. 2 Uncompressed Drug Products,Vol. 3 Liquid Products, Vol. 4 Semi-Solid Products, Vol. 5 Over theCounter Products, and Vol. 6 Sterile Products], CRC Press, Apr. 27,2004.

Useful solutions for oral or parenteral administration can be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, 18th ed. (MackPublishing Company, 1990). Formulations for parenteral administrationcan also include glycocholate for buccal administration,methoxysalicylate for rectal administration, or citric acid for vaginaladministration. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.Suppositories for rectal administration also can be prepared by mixingthe drug with a non-irritating excipient such as cocoa butter, otherglycerides, or other compositions which are solid at room temperatureand liquid at body temperatures. Formulations also can include, forexample, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, and hydrogenated naphthalenes. Formulations for directadministration can include glycerol and other compositions of highviscosity. Other potentially useful parenteral carriers for these drugsinclude ethylene-vinyl acetate copolymer particles, osmotic pumps,implantable infusion systems, and liposomes. Formulations for inhalationadministration can contain as excipients, for example, lactose, or canbe aqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or oily solutions foradministration in the form of nasal drops, or as a gel to be appliedintranasally. Retention enemas also can be used for rectal delivery.

Formulations of the present invention suitable for oral administrationcan be in the form of: discrete units such as capsules, gelatincapsules, sachets, tablets, troches, or lozenges, each containing apredetermined amount of the drug; a powder or granular composition; asolution or a suspension in an aqueous liquid or non-aqueous liquid; oran oil-in-water emulsion or a water-in-oil emulsion. The drug can alsobe administered in the form of a bolus, electuary or paste. A tablet canbe made by compressing or molding the drug optionally with one or moreaccessory ingredients. Compressed tablets can be prepared bycompressing, in a suitable machine, the drug in a free-flowing form suchas a powder or granules, optionally mixed by a binder, lubricant, inertdiluent, surface active or dispersing agent. Molded tablets can be madeby molding, in a suitable machine, a mixture of the powdered drug andsuitable carrier moistened with an inert liquid diluent.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients. Oral compositions preparedusing a fluid carrier for use as a mouthwash include the compound in thefluid carrier and are applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Itshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganismssuch as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyetheylene glycol), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as manitol, sorbitol,sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfilter sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Formulations suitable for intra-articular administration can be in theform of a sterile aqueous preparation of the drug that can be inmicrocrystalline form, for example, in the form of an aqueousmicrocrystalline suspension. Liposomal formulations or biodegradablepolymer systems can also be used to present the drug for bothintra-articular and ophthalmic administration.

Formulations suitable for topical administration, including eyetreatment, include liquid or semi-liquid preparations such as liniments,lotions, gels, applicants, oil-in-water or water-in-oil emulsions suchas creams, ointments or pastes; or solutions or suspensions such asdrops. Formulations for topical administration to the skin surface canbe prepared by dispersing the drug with a dermatologically acceptablecarrier such as a lotion, cream, ointment or soap. Useful are carrierscapable of forming a film or layer over the skin to localize applicationand inhibit removal. For topical administration to internal tissuesurfaces, the agent can be dispersed in a liquid tissue adhesive orother substance known to enhance adsorption to a tissue surface. Forexample, hydroxypropylcellulose or fibrinogen/thrombin solutions can beused to advantage. Alternatively, tissue-coating solutions, such aspectin-containing formulations can be used.

For inhalation treatments, inhalation of powder (self-propelling orspray formulations) dispensed with a spray can, a nebulizer, or anatomizer can be used. Such formulations can be in the form of a finepowder for pulmonary administration from a powder inhalation device orself-propelling powder-dispensing formulations. In the case ofself-propelling solution and spray formulations, the effect can beachieved either by choice of a valve having the desired spraycharacteristics (i.e., being capable of producing a spray having thedesired particle size) or by incorporating the active ingredient as asuspended powder in controlled particle size. For administration byinhalation, the compounds also can be delivered in the form of anaerosol spray from pressured container or dispenser which contains asuitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration also can be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants generally are known in the art, and include, forexample, for transmucosal administration, detergents and bile salts.Transmucosal administration can be accomplished through the use of nasalsprays or suppositories. For transdermal administration, the activecompounds typically are formulated into ointments, salves, gels, orcreams as generally known in the art.

The active compounds can be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. Liposomalsuspensions can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

Oral or parenteral compositions can be formulated in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit formrefers to physically discrete units suited as unitary dosages for thesubject to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and thetherapeutic effect to be achieved, and the limitations inherent in theart of compounding such an active compound for the treatment ofindividuals. Furthermore, administration can be by periodic injectionsof a bolus, or can be made more continuous by intravenous, intramuscularor intraperitoneal administration from an external reservoir (e.g., anintravenous bag).

Where adhesion to a tissue surface is desired the composition caninclude the drug dispersed in a fibrinogen-thrombin composition or otherbioadhesive. The compound then can be painted, sprayed or otherwiseapplied to the desired tissue surface. Alternatively, the drugs can beformulated for parenteral or oral administration to humans or othermammals, for example, in effective amounts, e.g., amounts that provideappropriate concentrations of the drug to target tissue for a timesufficient to induce the desired effect.

Where the active compound is to be used as part of a transplantprocedure, it can be provided to the living tissue or organ to betransplanted prior to removal of tissue or organ from the donor. Thecompound can be provided to the donor host. Alternatively or, inaddition, once removed from the donor, the organ or living tissue can beplaced in a preservation solution containing the active compound. In allcases, the active compound can be administered directly to the desiredtissue, as by injection to the tissue, or it can be providedsystemically, either by oral or parenteral administration, using any ofthe methods and formulations described herein and/or known in the art.Where the drug comprises part of a tissue or organ preservationsolution, any commercially available preservation solution can be usedto advantage. For example, useful solutions known in the art includeCollins solution, Wisconsin solution, Belzer solution, Eurocollinssolution and lactated Ringer's solution.

In conjunction with the methods of the present invention,pharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) can be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician canconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a drug as well as tailoringthe dosage and/or therapeutic regimen of treatment with the drug.

Generally, an effective amount of dosage of active compound will be inthe range of from about 0.1 to about 100 mg/kg of body weight/day, morepreferably from about 1.0 to about 50 mg/kg of body weight/day. Theamount administered will also likely depend on such variables as thetype of surgery or invasive medical procedure, the overall health statusof the patient, the relative biological efficacy of the compounddelivered, the formulation of the drug, the presence and types ofexcipients in the formulation, and the route of administration. Also, itis to be understood that the initial dosage administered can beincreased beyond the above upper level in order to rapidly achieve thedesired blood-level or tissue level, or the initial dosage can besmaller than the optimum.

Nonlimiting doses of active compound comprise from about 0.1 to about1500 mg per dose. Nonlimiting examples of doses, which can be formulatedas a unit dose for convenient administration to a patient include: about25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275mg, about 300 mg, about 325, about 350 mg, about 375 mg, about 400 mg,about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg,about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg,about 675 mg about 700 mg, about 725 mg, about 750 mg, about 775 mg,about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg,about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg,about 1050, mg, about 1075 mg, about 1100 mg, about 1125 mg, about 1150mg, about 1175 mg, about 1200 mg, about 1225 mg, about 1250 mg, about1275 mg, about 1300 mg, about 1325 mg, about 1350 mg, about 1375 mg,about 1400 mg, about 1425 mg, about 1450 mg, about 1475 mg, and about1500 mg. The foregoing doses are useful for administering the compoundsof the present invention according to the methods of the presentinvention.

As is understood by one of ordinary skill in the art, generally, whendosages are described for a pharmaceutical active, the dosage is givenon the basis of the parent or active moiety. Therefore, if a salt,hydrate, or another form of the parent or active moiety is used, acorresponding adjustment in the weight of the compound is made, althoughthe dose is still referred to on the basis of the parent or activemoiety delivered. As a nonlimiting example, if the parent or activemoiety of interest is a monocarboxylic acid having a molecular weight of250, and if the monosodium salt of the acid is desired to be deliveredto be delivered at the same dosage, then an adjustment is maderecognizing that the monosodium salt would have a molecular weight ofapproximately 272 (i.e. minus 1H or 1.008 atomic mass units and plus 1Na or 22.99 atomic mass units). Therefore, a 250 mg dosage of the parentor active compound would correspond to about 272 mg of the monosodiumsalt, which would also deliver 250 mg of the parent or active compound.Said another way, about 272 mg of the monosodium salt would beequivalent to a 250 mg dosage of the parent or active compound.

All percentages and ratios used herein, unless otherwise indicated, areby weight. The percent dimeric impurity is on an area percent basis,typically as quantified by analytical HPLC.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the inventionremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

Formulation Examples

Formulation for Intravenous Administration Ingredients AmountAntimicrobial Compound 0.1-1500 total mg Dextrose, USP 50 mg/ml Sodiumcitrate, USP 1.60-1.75 mg/ml Citric Acid, USP 0.80-0.90 mg/ml Water, USPq.s

This formulation for intravenous administration is formulated by heatingwater for injection to about 60° C. Next the sodium citrate, citric acidand dextrose are added and stirred until dissolved. A solution oraqueous slurry of the antimicrobial compound is added to the previousmixture and stirred until dissolved. The mixture is cooled to 25° C.with stirring. The pH is measured and adjusted if necessary. Lastly themixture is brought to the desired volume, if necessary, with water forinjection. The mixture is filtered, filled into the desired container(vial, syringe, infusion container, etc.), over wrapped and terminallymoist heat sterilized.

This formulation is useful for intravenous administration, either bolusor infusion, to a patient.

Tablets for Oral Administration Ingredients Per Tablet Per 4000 TabletsAntimicrobial Compound 0.1-1500 mg 0.4-6000 g Anhydrous Lactose, NF110.45 mg 441.8 g Microcrystalline 80.0 mg 320.0 g Cellulose NFMagnesium Stearate 1.00 mg 4.0 g Impalpable Powder NF CroscarmelloseSodium 2.00 mg 8.0 g NF Type AThe antimicrobial compound (any of the compounds equivalent to thedesired delivery strength, e.g., 50 to 1500 mg per tablet) is premixedwith ⅓ of the microcrystalline cellulose NF and ½ of the anhydrouslactose NF in a ribbon blender for 5 minutes at 20 RPM. To the premix isadded the remaining ⅔ of the microcrystalline cellulose NF and theremaining ½ of the anhydrous lactose NF. This is blended for 10 minutesat 20 RPM. Crosscarmellose sodium is added to the blended powders andmixed for 5 minutes at 20 RPM. Finally the magnesium stearate is addedto the mixture by passing through a 90 mesh screen and blended for anadditional 5 minutes at 20 RPM. The lubricated mixture is compressed toprovide tablets of 500 mg active ingredient.

These tablets are useful for oral administration to a patient.

Incorporation By Reference

The entire disclosure of each of the patent documents, includingcertificates of correction, patent application documents, scientificarticles, governmental reports, websites, and other references referredto herein is incorporated by reference in its entirety for all purposes.In case of a conflict in terminology, the present specificationcontrols.

Equivalents

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A process for preparing a quinolone compoundcomprising the step of reacting a des-chloro quinolone compound or apharmaceutically acceptable salt or ester thereof with a chlorinatingagent and an acid, wherein the molar ratio of the acid to des-chloroquinolone is from about 0.007 to about 0.02, wherein less than about0.40% of dimeric impurity on an area percent basis of the quinolone isproduced.
 2. A process according to claim 1 wherein the quinolonecompound is1-(6-Amino-3,5-difluoropyridin-2-yl)-8-chloro-6-fluoro-7-(3-hydroxyazetidin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid or a pharmaceutically acceptable salt or ester thereof and thedes-chloro quinolone compound is1-(6-amino-3,5-difluoropyridin-2-yl)-6-fluoro-7-(3-hydroxy-azetidin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid or a pharmaceutically acceptable salt or ester thereof.
 3. Aprocess according to claim 2 wherein the dimeric impurity is1-Amino-3-(azetidin-3-yloxy)-propan-2-ol-bis(N,N′-quinolone carboxylicacid), or a pharmaceutically acceptable salt or ester thereof.
 4. Aprocess according to claim 1 wherein the chlorinating agent isN-chlorosuccinimide.
 5. A process according to claim 1 wherein the acidis selected from the group consisting of sulfuric acid, hydrochloricacid, hydrobromic acid, phosphoric acid, trifluoroacetic acid, triflicacid, methanesulfonic acid, p-toluenesulfonic acid, or perchloric acid,and mixtures thereof.
 6. A process according to claim 1 wherein the acidis sulfuric acid.
 7. A process according to claim 1 where the reactionis run at a temperature from about 0° C. to about 30° C.
 8. A processaccording to claim 1 wherein the molar ratio of N-chlorosuccinimide todes-chloro quinolone is greater than about
 1. 9. A process according toclaim 1 using an acetate ester as a solvent.
 10. A process according toclaim 9 wherein said acetate ester is selected from the group consistingof methyl acetate, ethyl acetate, and mixtures thereof.
 11. A processaccording to claim 9 wherein said acetate ester is methyl acetate.
 12. Aprocess according to claim 1 comprising the further step of reacting thequinolone compound with a base.
 13. A process according to claim 12wherein the base is a hydroxide base.
 14. A process according to claim13 wherein the hydroxide base is selected from the group consisting ofsodium hydroxide, potassium hydroxide, lithium hydroxide, bariumhydroxide, and mixtures thereof.
 15. A process according to claim 14wherein the hydroxide base is potassium hydroxide.
 16. A processaccording to claim 12 using a mixture of isopropanol and water as asolvent.
 17. A process according to claim 1 wherein said process is acommercial scale process.
 18. A composition comprising the quinolonecompound1-(6-Amino-3,5-difluoropyridin-2-yl)-8-chloro-6-fluoro-7-(3-hydroxyazetidin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid or salt or ester thereof having less than about 0.40% of thecompound 1-Amino-3-(azetidin-3-yloxy)-propan-2-ol-bis(N,N′-quinolonecarboxylic acid), or a pharmaceutically acceptable salt or esterthereof.
 19. A composition according to claim 18 wherein saidcomposition is a commercial scale composition.
 20. The process of claim1 or 2, wherein the molar ratio of chlorinating agent to des-chloroquinolone is from about 1.05 to about 1.2.
 21. The process of claim 1 or2, wherein the molar ratio of chlorinating agent to des-chloro quinoloneis from about 1.04 to about 1.07.
 22. The process of claim 1 or 2,wherein the molar ratio of acid to des-chloro quinolone is from about0.008 to about 0.012.